improvement-bacillus-subtilis-spore-enumeration-label-analysis-flow

Improvement of <em>Bacillus subtilis</em> Spore Enumeration and Label Analysis in Flow Cytometry

The spores of Bacillus subtilis have already been proposed for different biotechnological and immunological applications; however, there is an increasing ...

FIOCRUZ Rondônia

The action of Cr-LAAO, an l-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on NADPH oxidase activation in isolated human neutrophil function was investigated. This enzyme has an intrinsic activity of hydrogen peroxide production. Cr-LAAO, in its native form, induces the ROS production in neutrophil and migration of cytosolic NADPH oxidase components p40, p47 and p67 to the membrane, and Rac, a GTPase protein member, with the involvement of intracellular signaling mediated by phospho PKC-α. In its inactive form, iCr-LAAO does not induce NADPH oxidase activation in neutrophil showing that the intrinsic enzymatic activity does not have a role in this process, suggesting that its primary structure is essential for the cell's stimulation. Accordingly, the data showed for the first time that the Cr-LAAO has a role in NADPH oxidase complex activation triggering relevant proinflammatory events in human neutrophils.

Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation.

BjcuL is a C-type lectin isolated from Bothrops jararacussu snake venom with specificity for binding β-d-galactose units. BjcuL is not toxic to human peripheral blood mononuclear cells (PBMCs), but it inhibits PBMC proliferation and stimulates these cells to produce superoxide anions and hydrogen peroxide primarily via lymphocyte stimulation; it does not stimulate the production of nitric oxide and PGE . The purpose of this study was to investigate the effect of BjcuL on PBMC activation with a focus on cytokine release modulating PBMC proliferation. The results showed for the first time that BjcuL coupled to FITC interacted with monocytes, B cells, natural killer (NK) cells, and with subpopulations of T cells. These cell-cell interactions can lead to cell activation and inflammatory cytokines release, such as IL-6 and TNF-α, as well as the anti-inflammatory cytokine IL-10. In addition, TNF-α release was attributed to NK cells and monocytes, whereas IL-10 was attributed to TCD4 and Treg cells when stimulated by BjcuL. The temporal cytokines profile produced by cells when stimulated with this lectin allows us to assert that BjcuL has immunomodulatory activity in this context.

Lectin isolated from Bothrops jararacussu venom induces IL-10 release by TCD4 cells and TNF-α release by monocytes and natural killer cells.

Snake venom phospholipases A (PLAs) have hemolytic, anticoagulant, myotoxic, oedematogenic, bactericidal, and inflammatory actions. BthTX-I, a Lys49-PLA isolated from  venom, is an example of Lys49-PLA that presents such actions. NLRP3 is a cytosolic receptor from the NLR family responsible for inflammasome activation via caspase-1 activation and IL-1β liberation. The study of NLRs that recognize tissue damage and activate the inflammasome is relevant in envenomation.

Inflammasome Activation Induced by a Snake Venom Lys49-Phospholipase A Homologue.

Cr-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, has been demonstrated as a potent stimulus for neutrophil activation and inflammatory mediator production. However, the mechanisms involved in Cr-LAAO induced neutrophil activation has not been well characterized. Here we investigated the mechanisms involved in Cr-LAAO-induced lipid body (also known as lipid droplet) biogenesis and eicosanoid formation in human neutrophils. Using microarray analysis, we show for the first time that Cr-LAAO plays a role in the up-regulation of the expression of genes involved in lipid signalling and metabolism. Those include different members of phospholipase A mostly cytosolic phospholipase A-α (cPLA-α); and enzymes involved in prostaglandin synthesis including cyclooxygenases 2 (COX-2), and prostaglandin E synthase (PTGES). In addition, genes involved in lipid droplet formation, including perilipin 2 and 3 (PLIN 2 and 3) and diacylglycerol acyltransferase 1 (DGAT1), were also upregulated. Furthermore, increased phosphorylation of cPLA-α, lipid droplet biogenesis and PGE synthesis were observed in human neutrophils stimulated with Cr-LAAO. Treatment with cPLA-α inhibitor (CAY10650) or DGAT-1 inhibitor (A922500) suppressed lipid droplets formation and PGE secretion. In conclusion, we demonstrate for the first time the effects of Cr-LAAO to regulate neutrophil lipid metabolism and signalling.

Cytosolic phospholipase A-α participates in lipid body formation and PGE release in human neutrophils stimulated with an L-amino acid oxidase from Calloselasma rhodostoma venom.

Bothropic venoms contains high amount of secreted phospholipases A (sPLAs) that play a significant role in leukocyte activation and inflammation. Monocytes and lymphocytes are highly functional immune system cells that mediate and provide efficient responses during the inflammation. NLRP3 inflammasome is a multiprotein complex found in immune system cells that is triggered by pathogen- and damage-associated molecular patterns, PAMPs and DAMPs, respectively. PLAs' effect on human peripheral blood mononuclear cells (PBMCs) is still incompletely understood. PBMCs were isolated by density gradient and incubated with RPMI (control), LPS, BthTX-I (PLA-Lys49) or BthTX-II (PLA-Asp49) isolated from Bothrops jararacussu venom, to evaluate viability, and the results showed that there was no cell death. RT-qPCR and immunoblot were used to assess the gene and protein expression of NLRP3 components. Results indicated that there was substantial amplification of ASC, Caspase-1, IL-6, and IL-1β in 1 h and NLRP3 in 2 h. Protein expression was measured, and the results revealed substantial expression of the NLRP3 inflammasome complex after 4 h. IL-1β and LDH was quantified in the supernatant of the cells. Taken together, the findings demonstrate that BthTX-I and BthTX-II activate the NLRP3 inflammasome complex in human PBMCs and contribute to the inflammatory response seen in envenoming.

NLRP3 inflammasome activation in human peripheral blood mononuclear cells induced by venoms secreted PLAs.

Photobiomodulation therapy associated with conventional antivenom treatment has been shown to be effective in reducing the local effects caused by bothropic venoms in preclinical studies. In this study, we analyzed the influence of photobiomodulation using light emitting diode (LED) on the oxidative stress produced by murine macrophages stimulated with  venom and it isolated toxins BthTX-I and BthTX-II. Under LED treatment, we evaluated the activity of the antioxidant enzymes catalase, superoxide dismutase, and peroxidase as well as the release of hydrogen peroxide and the enzyme lactate dehydrogenase. To investigate whether NADPH oxidase complex activation and mitochondrial pathways could contribute to hydrogen peroxide production by macrophages, we tested the effect of two selective inhibitors, apocynin and CCCP3, respectively. Our results showed that LED therapy was able to decrease the production of hydrogen peroxide and the liberation of lactate dehydrogenase, indicating less cell damage. In addition, the antioxidant enzymes catalase, superoxide dismutase, and peroxidase increased in response to LED treatment. The effect of LED treatment on macrophages was inhibited by CCCP3, but not by apocynin. These findings show that LED photobiomodulation treatment protects macrophages, at least in part, by reducing oxidative stress caused  venom and toxins.

Light Emitting Diode Photobiomodulation Enhances Oxidative Redox Capacity in Murine Macrophages Stimulated with  Venom and Isolated PLAs.

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 μg/mL, positive control) or Cr-LAAO (50 μg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1β and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1β release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1β was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

Bothrops venom contains a high amount of secreted phospholipase A (sPLAs) enzymes responsible for the inflammatory reaction and activation of leukocytes in cases of envenoming. PLAs are proteins that have enzymatic activity and can hydrolyze phospholipids at the sn-2 position, thereby releasing fatty acids and lysophospholipids precursors of eicosanoids, which are significant mediators of inflammatory conditions. Whether these enzymes have a role in the activation and function of peripheral blood mononuclear cells (PBMCs) is not known. Here we show for the first time how two secreted PLAs (BthTX-I and BthTX-II) isolated from the venom of Bothrops jararacussu affect the function and polarization of PBMCs. Neither BthTX-I nor BthTX-II exhibited significant cytotoxicity to isolated PBMCs compared with the control at any of the time points studied. RT-qPCR and enzyme-linked immunosorbent assays were used to determine changes in gene expression and the release of pro-inflammatory (TNF-α, IL-6, and IL-12) and anti-inflammatory (TGF-β and IL-10) cytokines, respectively, during the cell differentiation process. Lipid droplets formation and phagocytosis were also investigated. Monocytes/macrophages were labeled with anti-CD14, -CD163, and -CD206 antibodies to assay cell polarization. Both toxins caused a heterogeneous morphology (M1 and M2) on days 1 and 7 based on immunofluorescence analysis, revealing the considerable flexibility of these cells even in the presence of typical polarization stimuli. Thus, these findings indicate that the two sPLAs trigger both immune response profiles in PBMCs indicating a significant degree of cell plasticity, which may be crucial for understanding the consequences of snake envenoming.

Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLAs.

Lectins are a large group of proteins found in many snake venoms. BjcuL is a C-type lectin from Bothrops jararacussu snake venom that does not present cytotoxicity action on human peripheral blood mononuclear cells (PBMCs) at concentrations of 5 and 10 μg/mL. BjcuL demonstrates an immunomodulatory role in PBMCs with the production of pro- and anti-inflammatory cytokines (IL-2, IL-10, IFN-γ, IL-6, TNF-α, and IL-17) in addition to stimulate T cells to produce reactive oxygen species (ROS) that could play a role in the acute inflammatory reaction observed in the victims. Inflammasomes are an essential arm in cells of innate immunity to detect and sense a range of endogenous or exogenous, sterile, or infectious stimuli to elicit cellular responses and effector mechanisms. NLRP3 inflammasome is a significant target for this study, because the lectin is responsible for leukocyte activation stimulating the release of inflammatory mediators, which results in dynamic cellular responses to remove the detrimental process to the body in snakebites. Thus, this study aimed to investigate how isolated BjcuL from B. jararacussu venom affects NLRP3 inflammasome activation on PBMCs. For this, the cells were isolated by density gradient and incubated with BjcuL at different periods and concentrations for the evaluation of the activation of the NLRP3 inflammasome through gene and protein expressions of ASC, CASPASE-1, and NLRP3 by RT-qPCR, Western blot, and immunofluorescence, as well as the participation of Toll-like receptor 4 (TLR4) and ROS in the IL-1β production, a product resultant of the NLRP3 inflammasome activation. Herein, BjcuL interacts with TLR4 as demonstrated by in vitro and in silico studies and induces cytokines release via NF-κB signaling. By genic and protein expression assays, BjcuL activates NLRP3 inflammasome, and the pharmacological modulation with LPS-RS, an antagonist of TLR4; LPS-SM, an agonist of TLR4; MCC950, a specific NLRP3 inhibitor, and rotenone, an inhibitor of mitochondrial ROS, confirmed the participation of TLR4 and ROS in the NLRP3 inflammasome activation and IL-1β liberation. The effects of BjcuL on the regulation and activation of the NLRP3 inflammasome complex via TLR4 activation with ROS participation may be determinant for the development of the inflammatory local effects seen in snakebite victims. In addition, in silico together with in vitro studies provide information that may be useful in the rational design of TLR agonists as well as new adjuvants for immunomodulatory therapy.

Article	Year
Role of l-amino acid oxidase isolated from Calloselasma rhodostoma venom on neutrophil NADPH oxidase complex activation. Toxicon : official journal of the International Society on Toxinology\| PubMed ID: 29499246	2018
Lectin isolated from Bothrops jararacussu venom induces IL-10 release by TCD4 cells and TNF-α release by monocytes and natural killer cells. Journal of leukocyte biology\| PubMed ID: 31087703	2019
Inflammasome Activation Induced by a Snake Venom Lys49-Phospholipase A Homologue. Toxins\| PubMed ID: 31906173	2019
Cytosolic phospholipase A-α participates in lipid body formation and PGE release in human neutrophils stimulated with an L-amino acid oxidase from Calloselasma rhodostoma venom. Scientific reports\| PubMed ID: 32620771	2020
NLRP3 inflammasome activation in human peripheral blood mononuclear cells induced by venoms secreted PLAs. International journal of biological macromolecules\| PubMed ID: 35074331	2022
Light Emitting Diode Photobiomodulation Enhances Oxidative Redox Capacity in Murine Macrophages Stimulated with Venom and Isolated PLAs. BioMed research international\| PubMed ID: 35872869	2022
Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom. Life sciences\| PubMed ID: 36113732	2022
Phenotypic, functional and plasticity features of human PBMCs induced by venom secreted PLAs. Molecular immunology\| PubMed ID: 36812762	2023
A C-type lectin induces NLRP3 inflammasome activation via TLR4 interaction in human peripheral blood mononuclear cells. Cellular and molecular life sciences : CMLS\| PubMed ID: 37349530	2023

Charles Nunes Boeno

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